Site-specific incorporation of functional components into RNA by transcription using unnatural base pair systems

The creation of an extra, unnatural base pair that functions in replication, transcription, and translation, would provide a new system for the expansion of the genetic alphabet. In transcription, an unnatural base pair system could be used for making new RNA molecules containing functional components of interest at specific positions. We have developed several unnatural base pairs that can function in replication and transcription. Among them, the 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) pair and the Ds and pyrrole-2-carbaldehyde (Pa) pair exhibit high selectivity and efficiency in PCR amplification and T7 transcription, respectively. We performed PCR amplification involving the Ds-Px pair for DNA template preparation and T7 transcription involving the Ds-Pa pair with several modified Pa substrates for the incorporation of functional components, such as biotin- and fluorescein-linked Pa nucleotides, into RNA. This Ds-Px and Ds-Pa pair system could provide a powerful tool for the site-specific fluorescent labeling and immobilization of RNA molecules, as a detection system for RNA and RNA-protein interactions.